Name: reconstitution of nucleosomes with differentially isotope-labeled sister histones
Uploaded: 2017-03-26T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones at JoVE.com

作者感谢菲利普博士Selenko（FMP - 柏林）提供湿实验室空间和基础设施，通过研究补助经费的工作进行实验和德意志研究联合会（DFG）（LI 2402 / 2-1）。

1.核小体的重建与差异同位素标记（和非对称修改）组蛋白姐姐注:目前的协议描述了差异同位素标记的组蛋白H3核小体的重建。为此，组蛋白H3的两个池被使用;一个是15 N-标记的并含有在N-末端具有带6xHis标签，第二次是13 C标记，并含有在N-末端融合的链球菌肽（WSHPQFEK）。这两个变量从一个烟草蚀纹病毒（TEV）蛋白酶识别位点的本机H3序列分离。以制备额外不对称修饰的核，这两个H3池之一被包括在premodified形式。组蛋白池的修改前的可以由感兴趣同位素标记的组蛋白与相应的组蛋白修饰酶在必要的情况下为每种类型的反应的辅助因子/ PTM献血者1反应来进行6。各个PTM的有效放置可通过NMR光谱或质谱进行评估。组蛋白/ DNA的这里使用的量是粗糙的建议以获得具有10μm的近似浓度（如DNA浓度在260nm测得）。这最终产率是足以记录良好品质NMR谱与相对较短的采集时间核小体的制备。 <ol><li> 与差异同位素标记（和不对称修改）组蛋白H3组蛋白八聚体的重组 注:重组组蛋白可以毫克的量来生产在BL21（DE3）plysS中的细胞14。以获得的同位素标记的组蛋白，相同的表达/纯化协议之后，使用对细菌生长的含有15 NH 4 Cl和/或13 C-葡萄糖作为氮源和碳源的基本培养基外，对15 N-或13 C-标签respectively。纯化的组蛋白被广泛地透析5mM的β巯基乙醇和使用之前储存冻干。 <ol><li>溶解冻干组蛋白等分试样，含有〜5毫克每组蛋白在1mL去折叠缓冲液（7M胍盐酸，20毫摩尔Tris pH为7.5，10mM的DTT）。包括两种类型的组蛋白H3。吹打混匀，不要旋涡。保持冰管30分钟，使完全展开。 </li><li>通过测量280nm处的吸光度对展开缓冲器和使用与ExPASy门户的protParam工具计算的消光系数确定每一个组蛋白的确切浓度。 15 </li><li>混合核心组蛋白等摩尔比，牢记包括每两个组蛋白H3池的50％。通过使用较少的组蛋白集中的全部内容启动并相应地添加所有其他人。 </li><li>调整最终蛋白质浓度至约1毫克/毫升用折叠缓冲液中。 </li><li> Transfer中混合物至6 kDa的截止透析膜和透析针对1-2大号冷却重折叠缓冲液（10mM的Tris pH为7.5，2M氯化钠，1mM EDTA中，2毫摩尔DTT）于4℃的。改变缓冲至少一次透析已经进行了最少3小时后。执行最终透析O / N在4℃。 </li><li>收集透析材料，并在4℃下的最大速度在微量离心除去任何沉淀5分钟（通常无沉淀应观察）。通过测量280nm处的吸光度确定八聚体的浓度。在计算中，用组蛋白的添加消光系数，牢记双每个值。 </li><li>浓缩，使用10 kDa的分界离心过滤单元〜2毫升的最终体积。重新计算八聚体的浓度和估计的损失。在这一点上，50之间的八聚体浓度 - 应取得100μM的。 </li><li>凝胶过滤柱（在材料的表表示）连接到一个FPLC系统和平衡用的过滤微米并脱气以1mL /分钟的流速和0.5MPa的压力极限重折叠缓冲0.22 2柱体积（CV）。 注:凝胶过滤运行应在冷室中或在冷柜中进行。 </li><li>使用1毫升/分钟的流速注入浓缩材料，并收集1.0 - 1.5毫升馏分。 </li><li>按照色谱图，并在65〜观察组蛋白八聚体洗脱 - 68毫升。 注意:洗脱峰的小肩膀，并在〜80毫升的峰值分别代表自由H3-H4四聚体和H2A-H2B二聚体的存在。 </li><li>在18％的SDS-PAGE凝胶上运行每个收集的级分的20微升。 注:加载它们在凝胶上，以减少高盐浓度，因此，避免扭曲之前用水以至少2倍稀释的样品。这同样适用于在重折叠缓冲液样品的随后的SDS-PAGE分析。 </li><li>池相关FRA载有4组蛋白的凝胶染色的均匀分布判断，并确定浓度之前（步骤1.1.7）纯八聚体ctions。 </li><li> 1毫升Ni-NTA柱（ 材料数据表 ）连接到FPLC系统，用10 CV 0.22平衡微米过滤并脱气以1mL / min的流速重折叠缓冲液中。 注意:所述的Ni-NTA色谱应在冷室中或在冷柜中进行。 </li><li>使用1毫升/分钟的流速通过在Ni-NTA通过纯化的八聚体。 </li><li>收集流过，并保持样品SDS-PAGE / WB分析（分别为20μL和4μL）。接着，用10CV重折叠缓冲或直到达到基线信号的洗柱。 </li><li>用10CV复性使用1毫升/分钟的流速含有250mM咪唑缓冲液中洗脱。为保持SDS-PAGE / WB分析的样品（分别为20μL和4μL）。 </li><li>平衡的交流的1毫升亲和柱ommercial链霉（ 材料表 ）使用批处理/重力流设置含250 mM咪唑10 CV复性缓冲。 注:此色谱步骤应在冷室中进行。 </li><li>通过商业链霉亲和柱通过的Ni-NTA洗脱。 </li><li>通过收集流量和样本保持SDS-PAGE / WB分析（分别为20μL和4μL）。随后，用10CV复性缓冲。 </li><li>用10CV折叠含有2.5mM脱硫生物素缓冲液中洗脱。为保持SDS-PAGE / WB分析的样品（20和4μL分别）。 </li><li>测量八聚体浓度，添加量少10倍TEV蛋白酶和让反应在标准的塑料离心管中在4℃下继续过夜。 注:TEV的必需量，以获得完全消化（除去亲和标签的）依赖于原点（构建体）和活性（新鲜的，重组的TEV更活性相比在长时间储存​​所述一个 - 80℃）。尝试相比，八聚体（估计为蛋白质浓度）最初加入10倍的TEV少，并相应地调整。 </li><li>通过运行前的混合物的20微升和后TEV除了在一个18％的SDS-PAGE凝胶上检查有效消化。如果消化不完全，增加更多的TEV蛋白酶和再持续3-4小时孵化。 </li><li>透析针对使用50kDa的截止透析膜以除去TEV蛋白酶和调整缓冲组合物的重折叠缓冲液样品。 </li><li>使用10 kDa的分界离心过滤单元（从步骤1.1.7相同的过滤器单元，也可使用），以1.0-2.0毫升体积浓缩纯化不对称八聚体。测量最终浓度。要么使用后不久为核小体重构或调整到50％（体积/体积）甘油在-20℃保存更长时间。在这一点上与前面的步骤中期望的至少70％的损失，第Ë八聚体浓度应在20〜 - 50微米。 </li></ol></li><li> 核重建 <ol><li>如果八聚体被储存在50％甘油，在4℃下透析过夜针对与核小体重构继续之前重折叠缓冲液中。 </li><li>调整的DNA通过使用5 M氯化钠原液（含有核小体定位序列）的盐浓度至2M NaCl的。 注:将纯化的DNA，它包含或所需序列的多拷贝的质粒的纯化后或者分离通过PCR扩增合成应储存浓缩至〜在水中的50微米或标准的Tris-EDTA缓冲液。 </li><li>利用小规模试验确定最佳比例混合八聚体和DNA。使用重折叠缓冲液调整最终体积至〜3.0毫升。总是添加最后以防止混合的八聚体和在&lt;2M的盐浓度该DNA的任何可能性的八聚体。 <ol><li>执行初始小-SC强麦reconstitutions确定八聚体:通向最佳产量核DNA的比例。对于这一点，装配的0.9三种反应:1.0，1.0:1.0和1.1:1.0 DNA:八聚体比率微升50-100之间的音量。通常情况下，使用5微米的DNA的浓度。 </li><li>有如下（步骤1.2.4-1.2.8）中描述的重组进行，到了最后，通过测量260nm处的DNA浓度，并通过运行6％原生-PAGE DNA凝胶估计可溶性质量好的重组核小体量，用溴化乙锭，分别（ 图5A）染色。 </li></ol></li><li>混合转移至6 kDa的截止透析膜和透析针对1升，4℃过夜重折叠缓冲液中。 </li><li>从2减少透析缓冲液的盐浓度在一个逐步的方式，至0.85，0.64，和分别的0.2M NaCl洗涤。保持样品中的每个缓冲器至少3小时。有时沉淀在最后转换后形成的。在这种情况下Continue透析按计划和微量离心在下一步骤结束除去沉淀物。 </li><li>转印透析膜在烧杯用1L测定缓冲液（25mM的NaH 2 PO 4 / 的 Na 2 HPO 4 pH 6.8的25 mM氯化钠，2mM的DTT）中，并继续透析O / N在4℃下的。 </li><li>收集样品，除去任何沉淀物在最大速度步骤1.2.5通过离心在5分钟微量在4℃下形成，并使用30kDa的分界离心过滤单元来〜300微升的最终体积浓缩。 </li><li>测量DNA浓度在260nm，运行了18％的SDS-PAGE蛋白质凝胶（4微升的样品）和6％的天然-PAGE DNA凝胶（0.2μL样品），并存储样本在4℃几周。终浓度应在10-20微米的范围内。 </li></ol></li></ol>在两个姐妹组蛋白修饰反应2. NMR分析注:转让核小体组蛋白尾巴的二维1 H- 15 N相关光谱可以参考16，17，18被发现。此外，CH的分配X在2D的赖氨酸，丝氨酸和苏氨酸组1 H- 13 C相关光谱可参考16找到。 <ol><li> NMR设置和参考光谱的记录 注意:采用在测定缓冲液中的核小体样品的140微升酶测定法来进行到3毫米NMR管在与其他样品体积300K。不同的NMR管中，也可以使用。上述温度适宜，以获得高质量的核小体的组蛋白尾巴和良好的酶活性的1 H- 15 N相关光谱。 2D-梭华-Sofast-HMQC 1 H- 15 N和2D-HSQC 1 H- 13 C光谱带设置考勤分别录帽子赋予类似的采集时间。典型采集参数是128瞬变与1.024（1H）×128（15 N），复分和32瞬变与1.024（1H）×64（13℃）的两种不同类型的相关光谱的复杂分。对于二维光谱采集时间为〜30分钟。 <ol><li>置300 K作为在光谱仪样品温度。加10％的D 2 O中（体积/体积），以将用于分光计频率锁定的样品。 </li><li>放置在光谱仪样品和进行基本操作（锁定，调整垫片调整）。此外，确定最佳的脉冲长度和普通采集参数。 </li><li>记录一维和二维1 H- 15 N和1 H- 13 C参考光谱。 </li><li>过程参考光谱，并确保用于映射所述改性反应的信号被很好的解决，并具有良好的强度。 </li><li>恢复样品和我转移吨至一个离心管。 </li></ol></li><li> 酶促反应，NMR监测和定量分析的设置 <ol><li>复制并安排一系列交错的2D 1 H- 15 N和1 H- 13 C谱。瞄准的几个小时的总采集时间和基于来自该第一反应得到的酶促速率新的运行相应的调整。 </li><li>添加到样品的必要辅因子用于各个类型的修饰反应（1毫摩尔ATP / 2mM的MgCl 2的磷酸化，1mM的乙酰-CoA乙酰化，1mM的SAM为甲基， 等 ）。 </li><li>加的目的酶，通过移液混合，在光谱仪（快速执行这些操作）将样品转移回到在NMR管，并随后。 注意:如果不存在关于期望的酶活性的数据，调整所述衬底对酶的摩尔比为10:1。执行第一次试运行，并相应地调整实际运行。 </li><li>执行快速自动匀场，并使用参数的其余部分从参考光谱。 </li><li>启动一系列交错二维1 H- 15 N和1 H- 13 C谱。 </li><li>按照实时反应的进行，当反应达到完成，或者所需的水平停止采集。 </li><li>流程光谱与完全相同的参数之前。 </li><li>使用两种类型的相关光谱的不同习得顺序重复整个运行。如果在第一个实验中一个1 H 15 N个相关最早是在系列中，用1 H- 13 C相关现在开始。通过这一点，并具有相同的采集时间为两个光谱，能够绘制上，而在同一时间尺度两者差异同位素标记的组蛋白比较PTM反应。此外，有可能计算出平均值和情节误差棒。 注:thoroug该方法用于核磁共振信号分析和酶促反应的后续量化h的描述可以在这里找到19。 </li><li>选择从每个时间进程NMR谱该报告反应的进行的NMR信号，并计算使用用于NMR光谱的可视化和分析软件它们的相对信号强度。为此该报告的未经修改以及修改状态的信号。提取修改级别。 </li><li>绘制对反应时间修改级别的计算值。 </li></ol></li></ol>

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Chem. Int. Ed. 55 (29), 8262-8265 (2016).</li><li>St&#252;tzer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulations+of+DNA+Contacts+by+Linker+Histones+and+Post-translational+Modifications+Determine+the+Mobility+and+Modifiability+of+Nucleosomal+H3+Tails.">Modulations of DNA Contacts by Linker Histones and Post-translational Modifications Determine the Mobility and Modifiability of Nucleosomal H3 Tails.</a> Mol. Cell. 61 (2), 247-259 (2016).</li><li>Zhou, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H4+K16Q+Mutation,+an+Acetylation+Mimic,+Causes+Structural+Disorder+of+Its+N-terminal+Basic+Patch+in+the+Nucleosome.">Histone H4 K16Q Mutation, an Acetylation Mimic, Causes Structural Disorder of Its N-terminal Basic Patch in the Nucleosome.</a> J. Mol. Biol. 421 (1), 30-37 (2012).</li><li>Theillet, F. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+NMR+mapping+and+time-resolved+monitoring+of+serine+and+threonine+phosphorylation+in+reconstituted+kinase+reactions+and+mammalian+cell+extracts.">Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts.</a> Nat. Protoc. 8 (7), 1416-1432 (2013).</li><li>Lowary, P. T., Widom, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+DNA+sequence+rules+for+high+affinity+binding+to+histone+octamer+and+sequence-directed+nucleosome+positioning.">New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.</a> J. Mol. Biol. 276 (1), 19-42 (1998).</li><li>Hackenberger, C. P., Schwarzer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+ligation+and+modification+strategies+for+peptides+and+proteins.">Chemoselective ligation and modification strategies for peptides and proteins.</a> Angew. Chem. Int. Ed. 47 (52), 10030-10074 (2008).</li><li>Theillet, F. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+mapping+and+time-resolved+monitoring+of+lysine+methylation+by+high-resolution+NMR+spectroscopy.">Site-specific mapping and time-resolved monitoring of lysine methylation by high-resolution NMR spectroscopy.</a> J. Am. Chem. Soc. 134 (18), 7616-7619 (2012).</li><li>Lechner, C. C., Agashe, N. D., Fierz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traceless+Synthesis+of+Asymmetrically+Modified+Bivalent+Nucleosomes.">Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes.</a> Angew. Chem. Int. Ed. 55 (8), 2903-2906 (2016).</li><li>Bernstein, B. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bivalent+chromatin+structure+marks+key+developmental+genes+in+embryonic+stem+cells.">A bivalent chromatin structure marks key developmental genes in embryonic stem cells.</a> Cell. 125 (2), 315-326 (2006).</li></ol>

The author has nothing to disclose.

对于核重建，目前协议利用含有601代表智慧核小体定位序列20 165 bp的DNA模板，但类似的性能是使用的模板DNA不同长度的预期。该协议的设计和采用使用非对称类型的组蛋白H3。用同样的原理，也可以应用于该方法的其他核心组蛋白和另外可用于重构携带具有不同同位素标记两个不同的核心组蛋白复合物。该协议可以稍微还修改了最初重组蛋白亚复合物，而不是直接组蛋白八聚体?...

真核生物DNA在细胞核内的紧密包装成染色质。染色质的基本构建块是一个包含DNA的周围由四个核心组蛋白的两个副本（H3，H4，H2A，H2B）的八聚体复合物包裹的〜147碱基对的核小体核心颗粒。组蛋白窝藏翻译后后修饰过多。这些共价的取代，都直接通过影响该系统的物理化学和间接通过招募染色质重塑活动1，2，3诱导染色质结构的改变。通过这些手段，组蛋白翻译后修饰控制染色质的无障碍，因此，调节所有基于DNA的细胞功能4。 翻译后修饰是由组蛋白修饰酶系统主要是对核小体纳入核心组织相容的非结构化N末端段（反面）安装网元。由于对组蛋白尾部的相对短的序列的许多修饰位点，翻译后修饰是通过诱导或阻断后续修饰反应，被称为修饰串扰5的效果相互影响。因为核小体的整体对称结构的，改性反应和串扰机制被认为类似地发生在每个核小体组蛋白（姐姐组蛋白）的两个副本。这个概念最近被质疑和反驳其后。特别是，关于自由组蛋白H3尾部的肽和在核小体的体外酶测定法表明，一组H3激酶以不对称的方式6中引入磷酸。此外，基于亲和纯化的LC-MS / MS分析表明在几种类型的真核细胞7的不对称H3甲基化的核小体的存在。因此，非对称改性的核构成新种，并需要工具来揭开该控制它们的形成和分析串扰效应，这种不对称可能发挥的机制。 通常，免疫印迹（WB）和质谱（MS）分析已被用来检测组蛋白翻译后修饰。尽管其应用方便，WB从特异性/交叉反应问题的困扰。最重要的是，它不能执行同步多PTM分析和修饰反应8的直接定量。另一方面，MS分析采用了精密的仪器，需要高水平的训练，但可提供高特异性以及同时映射和量化多个翻译后修饰9。 &#160;然而，这两种方法都是破坏性和核小体配合物分析之前被离解，从而产生的组蛋白和/或组蛋白衍生的肽的混合物。这种操作删除分辨独立的能力上每两个姐姐组蛋白的发生，并报告该核小体组蛋白的特定复制修改状态修饰反应。 演变作为一种替代方法，以映射的PTM反应核磁共振（NMR）谱。核磁共振是无中断的，因此允许PTM事件的实时方式中重构的混合物的监视，甚至在完整细胞10,11。例程快速数据采集，以及用于高分辨率映射基于二维发展同位素标记（15 N和/或13 C）的样品的异质核相关方法12允许不同类型的翻译后修饰的的同时映射，如如丝氨酸/苏氨酸/酪氨酸磷酸化，赖氨酸乙酰化/甲基化，和精氨酸甲基13。根据PTM正在调查中，15 N或13 C-PR标签otocols可以用来标记充当变形记者蛋白质的官能团。因此，PTM映射可以通过以下&#39;感测&#39;上的化学环境的改变相应的功能基团的特性的化学位移的位移来执行。在大多数情况下，无论是NH和CH的化学基团可以被用来报告感兴趣的PTM的演变。 目前的协议描述了含有差异同位素标记的妹妹组蛋白核的产生。它结合NMR光谱法的灵活性同时使用1 H 15 N和1 H 13 C相关光谱具有不同的蛋白的亲和标签的利用所选重组组蛋白复合物的纯化映射翻译后修饰。值得注意的是，该协议采用了核重建一个特定的组蛋白的两个不同的池。这些池是差异同位素标记（一个<suP&gt; 15 N，其他与13℃），它们分别融合到多组氨酸和链霉亲和标签。以Ni-NTA和基于链霉亲和色谱串联亲和纯化方案最初使用Voigt 等 。 7被用来净化从对称的对应（ 图1A）的不对称物种。非对称组蛋白八聚体随后用于重建等效核小体复合物（ 图1B），使用标准的盐透析法14。此外，通过相同的方法，由具有组蛋白池之一预先改性，一PTM可以不对称结合到所得到的核小体。这些基板与组蛋白修饰酶和修改事件的后续核磁共振映射的反应使串扰机制表征无论是在顺式 （premodified组蛋白拷贝）和反式 （unmodifiED组蛋白复印件）（ 图1C）。

<table><tbody><tr><td>Guanidinium HCl</td><td>Applichem</td><td>A14199</td><td>Use high quality Gu-HCl</td></tr><tr><td>Tris&amp;nbsp;</td><td>Roth</td><td>4855.2</td><td></td></tr><tr><td>DTT</td><td>Applichem</td><td>A1101</td><td></td></tr><tr><td>NaCl</td><td>VWR chemicals</td><td>27810.364</td><td></td></tr><tr><td>EDTA</td><td>Roth</td><td>8043.2</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Applichem</td><td>A1046</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Applichem</td><td>A3902</td><td></td></tr><tr><td>Imidazole</td><td>Applichem</td><td>A1073</td><td></td></tr><tr><td>d-Desthiobiotin</td><td>Sigma</td><td>D1411</td><td></td></tr><tr><td>Ni-NTA Superflow</td><td>Qiagen</td><td>1034557</td><td></td></tr><tr><td>Strep-Tactin Superflow</td><td>IBA</td><td>2-1207-001</td><td></td></tr><tr><td>His-probe antibody</td><td>Santa Cruz</td><td>sc-8036</td><td></td></tr><tr><td>Strep-tactin conjugated HRP</td><td>IBA</td><td>2-1502-001</td><td></td></tr><tr><td>Hi-Load 16/600 Superdex 200 pg</td><td>GE Healthcare</td><td>28-9893-35</td><td></td></tr><tr><td>6 - 8 kDa dialysis membrane</td><td>Spectrumlabs</td><td>spectra/por 1, 132650</td><td></td></tr><tr><td>50 kDa dialysis membrane</td><td>Spectrumlabs</td><td>spectra/por 7, 132129</td><td></td></tr><tr><td>10 kDa centrigugal filter unit</td><td>Merck Millipore</td><td>UFC901024</td><td></td></tr><tr><td>30 kDa centrigugal filter unit</td><td>Merck Millipore</td><td>UFC903024</td><td></td></tr><tr><td>Solution-state NMR spectrometer&amp;nbsp;</td><td></td><td></td><td>at least 500 MHz operating frequency, equipped with a triple-resonance cryoprobe</td></tr><tr><td>&lt;strong&gt;Buffer name&lt;/strong&gt;</td><td>&lt;strong&gt;Component&lt;/strong&gt;</td><td></td><td></td></tr><tr><td>Unfolding Buffer</td><td>7 M Guanidinium HCl</td><td></td><td></td></tr><tr><td></td><td>20 mM Tris, pH 7.5</td><td></td><td></td></tr><tr><td></td><td>10 mM DTT</td><td></td><td></td></tr><tr><td>Refolding Buffer</td><td>10 mM Tris, pH 7.5</td><td></td><td></td></tr><tr><td></td><td>2 M NaCl</td><td></td><td></td></tr><tr><td></td><td>1 mM EDTA</td><td></td><td></td></tr><tr><td></td><td>2 mM DTT</td><td></td><td></td></tr><tr><td>Assay Buffer</td><td>25 mM Na&lt;sub&gt;2&lt;/sub&gt;H&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 6.8</td><td></td><td></td></tr><tr><td></td><td>25 mM NaCl</td><td></td><td></td></tr><tr><td></td><td>2 mM DTT</td><td></td><td></td></tr></tbody></table>

reconstitution of nucleosomes with differentially isotope-labeled sister histones

非对称修饰的核遏制饰有不同的集合翻译后后修饰的组蛋白的两个副本（妹妹组蛋白）。他们是新发现的物种，建立和功能影响未知的手段。目前的分析方法不足以检测的核小体组蛋白姐姐翻译后修饰的特定复制发生。这个协议提出了含差异同位素标记的妹妹组蛋白核小体在体外重构的生物化学方法。生成复杂的可也不对称修饰，组蛋白subcomplexes的折叠过程中包括组蛋白premodified池之后。这些非对称核制剂可以容易地与组蛋白修饰酶来研究使用核磁共振（NMR）谱由非对称预掺入PTM施加变形串扰机制反应。特别是，在修饰反应实时可以独立的两个姊妹组蛋白通过进行不同类型的核磁共振相关的实验中，对于各同位素类型量身定做进行映射。这种方法提供了手段来研究有助于不对称PTM方式对核小体复合物的形成和传播机制的串扰。

适当重折叠八聚体种类重构混合物的通过凝胶过滤柱运行（ 图2）后分离。含有三种不同类型的八聚体的重组八聚池经受串联亲和纯化方案。样品从所有的步骤中收集并通过SDS-PAGE和随后WB分析。其导致非对称物种的隔离的协议的正确执行的验证是通过以下的不同样品中两个亲和标签（的His-和Strep-）的存在（ 图3）来实现的。随后，将亲和标签被TEV...

Watch this Scientific Journal Video about Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones at JoVE.com

Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

这个协议描述了含有差异同位素标记的妹妹组蛋白核小体重构。同时，可使用premodified组蛋白拷贝之后产生不对称翻译后修饰的核小体。这些制剂可以很容易地用于研究修改串扰机制，同时在两个姐姐组蛋白，通过使用高分辨率核磁共振光谱。

核小体的重建与差异同位素标记的姐妹组蛋白

Asymmetrically modified nucleosomes contain the two copies of a histone (sister histones) decorated with distinct sets of Post-translational Modifications ...

reconstitution-nucleosomes-with-differentially-isotope-labeled-sister

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

10.6K 次观看.  Leibniz Institute of Molecular Pharmacology (FMP-Berlin). 这个协议描述了含有差异同位素标记的妹妹组蛋白核小体重构。同时，可使用premodified组蛋白拷贝之后产生不对称翻译后修饰的核小体。这些制剂可以很容易地用于研究修改串扰机制，同时在两个姐姐组蛋白，通过使用高分辨率核磁共振光谱。 

视频: Reconstitution of Nucleosomes with Differentially Isotope-labeled Sister Histones

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.

Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

科研

生物化学

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.&#160; Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

核糖体组蛋白的体外泛化和二次基质测定

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination ...

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi pyrrolysine tRNA-synthetase (MmAcKRS1) and its cognate tRNA (tRNAPyl) to assemble reconstituted mononucleosomes with site specific acetylated histones. MmAcKRS1 and tRNAPyl deliver AcK at an amber mutation site in the mRNA of choice during translation in Escherichia coli. This technique has been used extensively to incorporate AcK at H3 lysine sites. Pyrrolysyl-tRNA synthetase (PylRS) can also be easily evolved to incorporate other noncanonical amino acids (ncAAs) for site specific protein modification or functionalization. Here we detail a method to incorporate AcK using the MmAcKRS1 system into histone H3 and integrate acetylated H3 proteins into reconstituted mononucleosomes. Acetylated reconstituted mononucleosomes can be used in biochemical and binding assays, structure determination, and more. Obtaining modified mononucleosomes is crucial for designing experiments related to discovering new interactions and understanding epigenetics.

Site Specific Lysine Acetylation of Histones for Nucleosome Reconstitution using Genetic Code Expansion in Escherichia coli

Here we present a method to express acetylated histone proteins using genetic code expansion and assemble reconstituted nucleosomes in vitro.

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

Acetylated histone proteins can be easily expressed in Escherichia coli encoding a mutant, N&#949;-acetyl-lysine (AcK)-specific Methanosarcina mazi ...

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase &#946; and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

This protocol outlines in detail the preparation of nucleosomal complexes using two methods of sample preparation for freezing TEM grids.

制备与DNA修复因子络合的核小体核心颗粒用于冷冻电镜结构测定

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of ...

<meta charset="utf8"></head><body>用于单分子研究的原位核小体重构

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

<meta charset="utf8"></head><body>作者聚焦：用于单分子技术的高效核小体重构

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

用于单分子相关力和荧光显微镜的原位核小体组装

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

<meta charset="utf8"></head><body>用于组蛋白亚型富集和 PTM 表征的 2D 凝胶分离

Unveiling Histone Proteoforms using 2D-TAU Gel Electrophoresis

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which control nucleosome dynamics and determine cell fate. The nucleosome, which is the functional unit of chromatin, comprises DNA, four pairs of histones (H3, H4, H2A, and H2B) making up the globular core, and the linker histone H1, which stabilizes the chromatin structure. The amino (N)-terminal tails of the histones protrude from the globular core domains and undergo distinct PTMs that influence the chromatin landscape. Some evidence suggests that histone PTM homeostasis is crucial for preserving all physiological activities. The deregulation of histone PTMs is the primary cause of abnormal cellular proliferation, invasion, and metastasis. Therefore, developing methods for characterizing histone PTMs is crucial. Here, we describe an effective technique for isolating and analyzing histone isoforms. The method, based on the combination of two orthogonal separations, allows the enrichment of histone isoforms and the following mass spectrometry identification. The technique, originally described by Shechter et al., combines acid-urea polyacrylamide gels (TAU-GEL), which can separate basic histone proteins based on size and charge, and Sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), which can separate proteins by molecular weight. The result is a two-dimensional map of histone isoforms, suitable for in-gel digestion followed by mass spectrometry identification and western blot analysis. The result is a two-dimensional map of histone isoforms, suitable for both in-gel digestion followed by mass spectrometry identification and western blot analysis. This proteomic approach is a robust method that allows the enrichment of a single histone isoform and the characterization of new histone PTMs.

<meta charset="utf8"></head><body>作者聚焦:表观遗传修饰和代谢再连接作为癌症治疗的靶点

This method describes a simple and fast strategy for histone proteoform characterization and investigation.

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

Histones undergo various post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, acylation, and ubiquitination, which ...

核小体的重建与差异同位素标记的姐妹组蛋白

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原子力显微镜成像在核细胞结构和动力学研究中的应用

核糖体组蛋白的体外泛化和二次基质测定

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在埃斯切里西亚大肠杆菌

制备与DNA修复因子络合的核小体核心颗粒用于冷冻电镜结构测定

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

核小体的重建与差异同位素标记的姐妹组蛋白

摘要

探索更多视频

此视频中的章节

原子力显微镜成像在核细胞结构和动力学研究中的应用

核糖体组蛋白的体外泛化和二次基质测定

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在 埃斯切里西亚大肠杆菌

制备与DNA修复因子络合的核小体核心颗粒用于冷冻电镜结构测定

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

用于单分子相关力和荧光显微镜的原位核小体组装

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

使用 2D-TAU 凝胶电泳揭示组蛋白蛋白形式

网站特定赖氨酸乙酰化的组蛋白为核体重组使用遗传密码扩展在埃斯切里西亚大肠杆菌